CRISPR activation screen identifies TGFβ-associated PEG10 as a crucial tumor suppressor in Ewing sarcoma

As the second most common pediatric bone and soft tissue tumor, Ewing sarcoma (ES) is an aggressive disease with a pathognomonic chromosomal translocation t(11;22) resulting in expression of EWS-FLI1, an “undruggable” fusion protein acting as transcriptional modulator. EWS-FLI1 rewires the protein expression in cancer cells by activating and repressing a multitude of genes. The role and contribution of most repressed genes remains unknown to date. To address this, we established a CRISPR activation system in clonal SKNMC cell lines and interrogated a custom focused library covering 871 genes repressed by EWS-FLI1. Among the hits several members of the TGFβ pathway were identified, where PEG10 emerged as prime candidate due to its strong antiproliferative effect. Mechanistic investigations revealed that PEG10 overexpression caused cellular dropout via induction of cell death. Furthermore, non-canonical TGFβ pathways such as RAF/MEK/ERK, MKK/JNK, MKK/P38, known to lead to apoptosis or autophagy, were highly activated upon PEG10 overexpression. Our study sheds new light onto the contribution of TGFβ signalling pathway repression to ES tumorigenesis and suggest that its re-activation might constitute a novel therapeutic strategy.

The Supplementary Table S1 contains LIBerty gene list and rankings. The  Supplementary Table S2 contains LIBerty gRNA list and rankings. The Supplementary  Table S3 contains gRNA 1 sequences from the LIBerty library. The Supplementary  Table S4 contains list of antibodies used in this study. The Supplementary Table S5 contains list of primers used in this study. Tables S1-S3 and S5 as separate Excel files.

Supplementary Figures
Supplementary Figure S1. Quality check of the LIBerty library in plasmid format.
A, NGS analysis of the plasmid LIBerty library for frequency of counts per gRNA. B, Comparison of the suggested guidelines for custom libraries to LIBerty library data.

Supplementary Figure S2. Correlation of screen replicates.
A, Correlation of NGS data points within samples collected on day three post transduction used for further analysis as control or time point zero. B, Correlation of NGS data points within samples collected on day ten post transduction used for further analysis as time point one. C, Correlation of NGS data points within samples collected on day twenty post transduction used for further analysis as time point two. All coefficients were calculated by PinAPL-Py pipeline. Image was created by using PinAPL-Py version 2.8.1, pinapl-py.ucsd.edu. NGS analysis of the CRISPRa screen was done via PinAPL-Py with gRNA counts as main readout. A-B, gRNA counts of each unique gRNA from time point one compared to time point zero with non-targeting gRNAs in orange, AAVS1 and positive controls labeled according to the legend. C-D, gRNA counts of each unique gRNA from time point two compared to time point zero with non-targeting gRNAs in orange, AAVS1 and positive controls labeled according to the legend. All subfigures were created by using GraphPad version 8, www.graphpad.com.

Supplementary Figure S4. Cell numbers decline in SKNCM SAM clonal cell lines overexpressing PEG10.
A, Quantification of the number of PEG10 overexpressing SKNMC SAM cells with the seeding amount highlighted via the dotted line. Image was created by using GraphPad version 8, www.graphpad.com.

Supplementary Figure S6. Replicates of the signaling investigation of the TGFβ pathways and apoptotic pathway in TGFBR2 and PEG10 overexpressing SKNMC SAM cells.
A, Biological replicates of the Western Blot measuring PEG10, TGFBR2 protein levels, cleavage status of PARP, Caspase 3 and 7, phosphorylation status of ERK, JNK, P38, AKT and SMAD2 in TGFBR2 and PEG10 overexpressing SKNMC SAM cells 4 days post transduction. Image was created by using Image Lab version 6.1, www.biorad.com/en-ch/product/image-lab-software. B, C, Densitometry of the Western Blot measuring PEG10, TGFBR2 protein levels, cleavage status of PARP, Caspase 3 and 7, phosphorylation status of ERK, JNK, P38, AKT and SMAD2 in TGFBR2 and PEG10 overexpressing SKNMC SAM cells 4 days post transduction. β-tubulin was used to normalize relative protein expression of PEG10, TGFBR2, Caspase 3 and 7. Cleaved PARP was normalized with total length PARP. Phosphorylated ERK1/2, SAPK/JNK, P38, AKT and SMAD2 were normalized with total ERK1/2, SAPK/JNK, P38, AKT and SMAD2. Image was created by using GraphPad version 8, www.graphpad.com. D, Biological replicate of the Western Blot measuring cleavage status of Caspase 8 and 9 in TGFBR2 and PEG10 overexpressing SKNMC SAM cells 4 days post transduction. Image was created by using Image Lab version 6.1, www.bio-rad.com/en-ch/product/image-lab-software. E, Densitometry of the Western Blot measuring cleavage status of Caspase 8 and 9 in TGFBR2 and PEG10 overexpressing SKNMC SAM cells 4 days post transduction. Cleaved Caspase 8 and 9 were normalized with total length Caspase 8 and 9. Image was created by using GraphPad version 8, www.graphpad.com.

Supplementary Figure S7: Original Western blots used in this study.
Most possible uncropped images of Western blots used in main and supplementary figures. Targets for used antibodies are indicated on the left from the bands. Areas of the blot used in Figures are highlighted by orange delineation. All images were created by using Image Lab version 6.1, www.bio-rad.com/en-ch/product/image-lab-software.